JOURNAL OF NATURAL HISTORY, 2016 http://dx.doi.org/10.1080/00222933.2016.1193643 Morphology of tube-like threads related to Limnochares aquatica (L., 1758) (Acariformes: Hydrachnidia: Limnocharidae) in the laboratory A.B. Shatrova, E.V. Soldatenkob and O.V. Gavrilovac aZoological Institute, Russian Academy of Sciences, St. Petersburg, Russia; bDepartment of Biology, Smolensk State University, Smolensk, Russia; cDepartment of Microbiology, St. Petersburg State University, St. Petersburg, Russia ABSTRACT ARTICLE HISTORY Water mites Limnochares aquatica (L., 1758) during maintenance in Received 9 April 2015 the laboratory for a long period of time in constant conditions Accepted 20 May 2016 fl periodically produced certain whitish occulent material consist- KEYWORDS ing of long rigid unbranched tube-like threads 1.3 ± 0.3 µm in Water mites; tube-like diameter crossing freely. These threads were studied using light- threads; TEM; SEM; optical as well as transmission electron microscopical and scan- Limnochares aquatica ning electron microscopical methods. Microbiological staining was also applied to the threads to exclude their bacterial or fungal origin. The thread wall is built of fine fibrils arranged at different angles to the long axis of threads that is reflected in a certain stratification of the wall. Threads are mostly hollow or may contain electron-dense homogeneous material. No cell components are present in the thread composition. Numerous dermal glands with their small slit-like orifice scattered throughout the mite body surface are thought to produce these threads. Most probably the thread formation is a reaction of mites to stress under laboratory conditions, and this is expected to be a type of defensive reaction. Introduction Production of protein threads known as silk is widely distributed among arthropods, in particular spiders (Stubbs et al. 1992;Foelix1996; Vollrath et al. 1996;Craig1997, 2003; Downloaded by [University of Cambridge] at 00:01 09 August 2016 Vollrath 2000;DeBakkeretal.2006;Sponneretal.2007;Krafft and Cookson 2012; Blackledge 2013), pseudoscorpions (Hunt 1970;Schaller1971;Kovoor1987;Annamalai and Jayaprakash 2012), mites (Wallace and Mahon 1972; Alberti and Ehrnsberger 1977; Bolland 1983;Gerson1985; Manson and Gerson 1996; Alberti and Coons 1999) and insects (Schaller 1971; Kovoor and Zylberberg 1980; 1982; Kristensen 2003;Kebedeetal.2014; etc.). Organs involved in silk formation are thought to have developed several times and independently in every group (Craig 2003) and are localised either de novo on the abdominal region, as in spiders and some pseudoscorpions (Schaller 1971), or are trans- formed from prosomal glands, as in some pseudoscorpions and mites (Gerson 1985). CONTACT A.B. Shatrov [email protected] Zoological Institute, Russian Academy of Sciences, Universitetskaya emb. 1, 199034, St. Petersburg, Russia © 2016 Informa UK Limited, trading as Taylor & Francis Group 2 A. B. SHATROV ET AL. Among acariform mites, spinning ability is characterised for representatives of several terrestrial families, such as Tetranychidae, Eriophyidae, Camerobiidae, Cunaxidae and Bdellidae (Alberti and Coons 1999), providing a great diversity of important functions (Witte 1991; Alberti and Coons 1999; Clotuche et al. 2011; Kanazawa et al. 2011;LeGoff et al. 2011; Fernandez et al. 2012; Yano 2012; etc.). It is accepted that in mites, especially in Tetranychidae (Gerson 1985; Alberti and Coons 1999), as well as in preudoscorpions (Kovoor 1987; Annamalai and Jayaprakash 2012), in contrast to other arachnids, produc- tion of silk is realised by the reorganised salivary glands (Gerson 1985). By contrast to this supposition, it was shown that in representatives of the pseudoscorpion genus Serianus, males spin their signalling threads ‘with the opistosomal spinning glands which open at the anus’ (Schaller 1971, p. 426). In eriophyoid mites, spinning organs were not identified (Manson and Gerson 1996) although webbing around mites’ colonies of some species is found to be enormous. In the highly diverse water mites (Hydrachnidia), males of several families with indirect sperm transfer produce so-called ‘guiding/signalling threads’, supporting sper- matophores, by special glands of the genital tract (Witte 1984, 1991; Proctor 1992; Alberti and Coons 1999; Witte and Döring 1999). A similar strategy is also found in representatives of some terrestrial families of Parasitengona (Proctor et al. 2015). No other data on silk organs, or on silk formation, were obtained for the whole history of water mite observations. In previous work (Shatrov 2014;Shatrovetal.2014), we have found that several water mite species appear to produce various amounts of silk during maintaining in the laboratory. This silk consisted of long, rigid, unbranched, hollow, tube-like threads varying from 700 nm to 2.5 µm in diameter. Transmission electron micro- scope (TEM) examination of silk threads of Limnesia undulata (O.F. Müller) revealed that their walls are composed of fine fibrous material of different density and orientation. The threads’ lumen did not contain cell membrane or any cell compo- nents, although variously organised non-cellular core may be present (Shatrov 2014; Shatrov et al. 2014). Specific staining revealed neither DNA nor microbial walls in threads’ composition, so the microbial origin of threads was excluded. The observed silk formation did not correspond to the mite reproduction behaviour because it lasted from late summer until mid autumn when the mites had already completed detachment of both eggs and spermatophores. It was supposed that dermal glands, Downloaded by [University of Cambridge] at 00:01 09 August 2016 an evolutionary acquisition of water mites, may be involved in the silk secretion (Shatrov 2013). We started keeping Limnochares aquatica (L.) in the laboratory, hoping to acquire additional data on the phenomenon of silk production by water mites. Generally, L. aquatica is a Holarctically distributed species with a well-known biology (Böttger 1972). Moreover, secretion supposedly of their dermal glands was biochemically tested, and behavioural experiments were performed with regard to the relationship of these mites with their potential natural enemies – predatory fishes (Kirstein and Martin 2009, 2010). It was shown that the secreted protein has a molecular weight of 30 kDa with additional components of a low weight. It was also demonstrated that fishes spat out both swallowed mites and food saturated with their secretion (Kirstein and Martin 2009, 2010), a behaviour which was also revealed earlier for some other species (Kerfoot 1982; Proctor and Garga 2004). On the other hand, in some Russian northern rivers, JOURNAL OF NATURAL HISTORY 3 the stomachs of fishes were found filled with red water mites of uncertain species (Sokolov 1940). The main purpose of the present study is a detailed morphological examination of threads secreted by water mites L. aquatica during their maintenance in the laboratory. Materials and methods Mites, collecting site and laboratory maintenance Several adult mites of L. aquatica were captured 19 September 2014 in the artificial lake Dubrovenskoye (54°47ʹN, 31°56ʹE) – the widest part of the same river, the right tributary of the river Dnepr in the north vicinity of Smolensk city (around 9 km from the city centre). Three specimens of these mites – two males and one female (Tuzovskiy 1979) – were taken for the experiment. The remaining mites were also kept in the laboratory for control observations, but were out of the strict experiment, subject to only periodic examination. Three experimental specimens were placed separately in the same plastic 100-mL container with a pure bottled artesian water (certification of conformity N POCC RU. AE05.H02957, www.smolvoda.ru) distributed in Smolensk city. The containers were sterilised using hot steam, and afterwards during usage they were kept loosely covered with a small Petri dish to allow the intake of air. Approximately once a week, we changed the water in the containers for fresh water, and the containers were newly sterilised. The experiment continued until March 2015. For the whole time of the experiment, the mites did not feed and were active. The males died 15 May and 30 October 2015 after they moulted in January–February 2015. The female was still living up to January 2016, did not moult and periodically produced some amount of silk. During the experiment, the mites were active during the day and inactive at night. The water was kept absolutely free of plants, plant leaves and any other additional materials and substances. The containers were constantly maintained at a temperature of 20–21°С, and with natural lightning. The containers were examined daily or once every 2 days. To take photographs, the mites were replaced for a short time into a Petri dish, allowing a good visualisation. Photographs were taken with a Canon G11 digital camera with a resolution of 10 Mpx. Mites used for scanning electron microscope (SEM) study (five specimens) were Downloaded by [University of Cambridge] at 00:01 09 August 2016 captured 12 July 2009 in Lake Maloye Strechnoye (55°51ʹN, 31°77ʹE, Smolensk Province, Demidovskiy District, National Park ‘Smolenskoye Poozerye’, around 4 km from Przhevalskoye town) and were nearly immediately fixed. Light-microscope observation For the light-microscope observation, the entire wisp or boll of threads or their smaller portions were removed from containers using a microscopic needle. Both temporary water and constant dry preparations